Worm Breeder's Gazette 14(1): 48 (October 1, 1995)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
School of Biology & Biochemistry University of Bath Bath BA2 7AY U.K.
The recently cloned avermectin receptor consists of a glutamate sensitive beta subunit and an avermectin sensitive alpha subunit which when co-expressed in Xenopus oocytes lead to the formation of avermectin potentiated glutamate gated chloride ion channels(1). Leon Avery et al have suggested that this receptor mediates the inhibition of pharyngeal muscle contraction via the glutamatergic motor neuron M3(2). Screening of the YAC polytene filter and subsequent cosmids with the 5' end of the beta subunit localised the gene and controlling 5' sequences to cosmid C35E8, which maps to chromosome 1. This cosmid was used as the template for PCR amplification of a DNA fragment consisting of 1426 bp of 5' sequence before the start methionine, plus sequence encoding the first 24 amino acids of the subunit. The fragment was subcloned into the LacZ expression vector, pPD22.11(3) to form a translational fusion. After worm injections we established 6 stably transformed lines which all showed the same beta-galactosidase activity. X-gal staining was present in the 3 pairs of pm4 pharyngeal muscle cell nuclei. No other nuclei consistently stained for X-gal. We have not looked closely at the developmental expression of this subunit but we did see pharyngeal staining in some larval stages (L2/3 onwards) and stained nuclei were seen in some developing eggs. These results provide further evidence that the Glu-Cl receptor mediates the glutamatergic inhibition of pharyngeal muscle via the M3 motor neuron, and point to inhibition of pharyngeal pumping as a major mode of action for avermectin. It must be remembered however that these experiments have localised the glutamate-binding beta subunit only. The avermectin binding alpha subunit may be expressed in a number of locations , for example body wall muscle, as well as pharyngeal muscle. Acknowledgements We thank Ian Hope for his help with the construction of transgenic worms and Donna Albertson for helping us in the identification of stained nuclei. References (1) Cully et al. (1994) Nature 371: 707-711. (2) Avery et al. (1995) WBG 13(4): 72-73. (3) Fire et al. (1990) Gene 93: 189-198.